Mechanisms Of Cell Cycle Progression Research Articles

Nimbin (N1) and analog N3 from the neem seeds suppress the migration of osteosarcoma MG-63 cells and arrest the cells in a quiescent state mediated via activation of the caspase-modulated apoptotic pathway.

Natural products are considered effective sources for new therapeutic research and development. The numerous therapeutic properties of natural substances in traditional medicine compel us to investigate the anti-cancer properties of Nimbin (N1) and its semi-natural analog Nimbic acid (N3) from Azadirachta indica against MG-63 Osteosarcoma cells. The therapeutic efficacy of N1 and N3 were screened for their toxicity and cytotoxic activity using L6 myotubes, zebrafish larvae and MG-63 osteosarcoma cells. The mitochondrial membrane potential was evaluated using the Rhodamine 123 stain. Further, the nuclear and cellular damage was distinguished using Hoechst and Acridine orange/EtBr stain. The mechanism of cell cycle progression, cellular proliferation and caspase cascade activation was screened using scratch assay, flow cytometry, and mRNA expression analysis. The Nimbin and analogue N3 were found to be non-toxic to normal L6 cells (Rat skeletal muscles), exhibited cytotoxicity in MG-63 cells, and were exposed to be an active inhibitor of cell proliferation and migration. Analogs N1 and N3 induced negative mitochondrial membrane potential when stained with Rhodamine 123, leading to nuclear damage and apoptosis stimulation using AO/EtBr and Hoechst. Further, N1 and N3 induced cell cycle arrest in G0/G1 phase in flow cytometry using PI staining and induced apoptosis by activating the caspase cascade and upregulated Caspase 3 and caspase 9. The study demonstrated cytotoxic activity against MG-63 osteosarcoma cells while being non-toxic to normal L6 cells. These compounds inhibited cell proliferation and migration, induced mitochondrial dysfunction, nuclear damage, and apoptosis stimulation. Furthermore, N1 and N3 caused cell cycle arrest and activated the caspase cascade, ultimately leading to apoptosis. These findings indicate that N1 and N3 hold promise as potential candidates used alone or combined with existing drugs for further investigation and development as anti-cancer agents.

Abstract 1427: NQO1/c-Fos/CKS1 signaling pathway regulates cell cycle progression at the G2/M phase

Abstract Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is associated with tumor cell proliferation and growth in several human cancer types. However, the molecular mechanisms underlying the activity of NQO1 in cell cycle progression are currently unclear. Here, we report a novel function of NQO1 in modulation of cyclin-dependent kinase subunit (CKS1), a cell cycle regulator, at the G2/M phase, through effects on the stability of c-Fos. We analyzed the roles of NQO1/c-Fos/CKS signaling pathway in cell cycle progression in cancer cells using synchronization of cell cycle and flow cytometry. The mechanisms of NQO1/c-Fos/CKS1-mediated regulation of cell cycle progression in cancer cells were studied by siRNA, overexpression system, reporter assay, co-immunoprecipitation, pull-down assay, microarray analysis, and CDK1 kinase assay. In addition, publicly available data sets and immunohistochemistry were used to investigate the correlation between NQO1 expression levels and clinicopathological features in cancer patients. Our results suggest that NQO1 directly interacts with the unstructured DNA-binding domain of c-Fos implicated in proliferation, differentiation, development and survival of cancer and inhibits its proteasome-mediated degradation, thereby inducing CKS1 expression and regulating cell cycle progression at the G2/M phase. Notably, NQO1 deficiency in human cancer cell lines led to suppression of c-Fos-mediated CKS1 expression and cell cycle progression. Consistent with this finding, high NQO1 expression was correlated with increased CKS1 and poor prognosis in cancer patients. Our collective results support a novel regulatory role of NQO1 in the mechanism of cell cycle progression at the G2/M phase through effects on c-Fos/CKS1 signaling pathway in cancer. Citation Format: Eun-Taex Oh, Ha Gyeong Kim, Yunmi Cho, Heon Joo Park. NQO1/c-Fos/CKS1 signaling pathway regulates cell cycle progression at the G2/M phase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1427.

NQO1 regulates cell cycle progression at the G2/M phase.

Rationale: Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is associated with tumor cell proliferation and growth in several human cancer types. However, the molecular mechanisms underlying the activity of NQO1 in cell cycle progression are currently unclear. Here, we report a novel function of NQO1 in modulation of the cell cycle regulator, cyclin-dependent kinase subunit-1 (CKS1), at the G2/M phase through effects on the stability of c‑Fos. Methods: The roles of the NQO1/c-Fos/CKS1 signaling pathway in cell cycle progression were analyzed in cancer cells using synchronization of the cell cycle and flow cytometry. The mechanisms underlying NQO1/c-Fos/CKS1-mediated regulation of cell cycle progression in cancer cells were studied using siRNA approaches, overexpression systems, reporter assays, co-immunoprecipitation, pull-down assays, microarray analysis, and CDK1 kinase assays. In addition, publicly available data sets and immunohistochemistry were used to investigate the correlation between NQO1 expression levels and clinicopathological features in cancer patients. Results: Our results suggest that NQO1 directly interacts with the unstructured DNA-binding domain of c-Fos, which has been implicated in cancer proliferation, differentiation, and development as well as patient survival, and inhibits its proteasome-mediated degradation, thereby inducing CKS1 expression and regulation of cell cycle progression at the G2/M phase. Notably, a NQO1 deficiency in human cancer cell lines led to suppression of c-Fos-mediated CKS1 expression and cell cycle progression. Consistent with this, high NQO1 expression was correlated with increased CKS1 and poor prognosis in cancer patients. Conclusions: Collectively, our results support a novel regulatory role of NQO1 in the mechanism of cell cycle progression at the G2/M phase in cancer through effects on c‑Fos/CKS1 signaling.

An evaluation of palbociclib as a breast cancer treatment option: a current update

ABSTRACT Introduction Patients with hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer have benefitted from treatment with palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor capable of selectively targeting mechanisms of cell cycle progression that contribute to tumor cell proliferation. Palbociclib use in this setting demonstrates improved progression-free survival when given in combination with aromatase inhibitors or fulvestrant. Areas covered The authors describe the current state of research surrounding palbociclib use in breast cancer, present evidence supporting a role for palbociclib in additional subtypes of metastatic breast cancer such as HER2-positive (HER2+) and triple-negative, report ongoing clinical trials aimed at expanding the scope of use for palbociclib, and discuss expected clinical results that will better inform decisions on including palbociclib as a part of breast cancer treatment strategies. Expert opinion Preclinical and clinical studies have shown promising evidence for palbociclib use in metastatic HER2+ and androgen receptor-expressing triple-negative breast cancer but mixed results in the adjuvant/neoadjuvant setting, where differences may only be detectable in high-risk disease. Palbociclib combinations may constitute viable replacements for chemotherapy in the neoadjuvant setting as part of de-escalation strategies. Investigation into synergy of palbociclib with immunotherapies is also ongoing based on non-canonical effects of CDK4/6 inhibition on the tumor immune microenvironment.

Non-destructive, label free identification of cell cycle phase in cancer cells by multispectral microscopy of autofluorescence

BackgroundCell cycle analysis is important for cancer research. However, available methodologies have drawbacks including limited categorisation and reliance on fixation, staining or transformation. Multispectral analysis of endogenous cell autofluorescence has been shown to be sensitive to changes in cell status and could be applied to the discrimination of cell cycle without these steps.MethodsCells from the MIA-PaCa-2, PANC-1, and HeLa cell lines were plated on gridded dishes and imaged using a multispectral fluorescence microscope. They were then stained for proliferating cell nuclear antigen (PCNA) and DNA intensity as a reference standard for their cell cycle position (G1, S, G2, M). The multispectral data was split into training and testing datasets and models were generated to discriminate between G1, S, and G2 + M phase cells. A standard decision tree classification approach was taken, and a two-step system was generated for each line.ResultsAcross cancer cell lines accuracy ranged from 68.3% (MIA-PaCa-2) to 73.3% (HeLa) for distinguishing G1 from S and G2 + M, and 69.0% (MIA-PaCa-2) to 78.0% (PANC1) for distinguishing S from G2 + M. Unmixing the multispectral data showed that the autofluorophores NADH, FAD, and PPIX had significant differences between phases. Similarly, the redox ratio and the ratio of protein bound to free NADH were significantly affected.ConclusionsThese results demonstrate that multispectral microscopy could be used for the non-destructive, label free discrimination of cell cycle phase in cancer cells. They provide novel information on the mechanisms of cell-cycle progression and control, and have practical implications for oncology research.

Abstract P4-07-07: Triple targeted combination therapy with tucatinib, palbociclib and fulvestrant is active in hormone receptor and HER2-positive human breast tumor cell lines

Abstract Hormone receptor and HER2-positive (HR+/HER2+) breast cancer displays increased resistance to anti-hormonal and HER2-targeted agents. HER2 and estrogen receptor signaling converge at cyclin D1 and CDK4/6 complex, resulting in the increased cyclin D1 expression and accelerated progression of the cell cycle. Therefore, triple blockade of the estrogen receptor, HER2 and CDK4/6 is reasonable as a novel approach to treatment of HR+/HER2+ disease. Here for the first time we report high activity of novel combination therapy with HER2-targeted small molecule inhibitor tucatinib, CDK4/6 inhibitor palbociclib, and selective estrogen receptor blocker fulvestrant in human breast tumor cell lines. Methods: HR+/HER2+ human breast tumor cell lines BT474, MDA-MB-361 and UACC-812 were cultured in standard conditions. The subclones of BT474 and MDA-MB-361 resistant to tucatinib and palbociclib were generated by culturing cells in the increasing concentrations of inhibitors over 6 months. Cell survival assays were performed with Cell Titer Glow (Promega, Madison, WI) after 72 hrs of treatment with vehicle, single agents tucatinib, palbociclib and fulvestrant, and with dual and triple combinations. Tucatinib was provided by Seattle Genetics (Seattle, WA), palbociclib and fulvestrant were purchased from Selleckchem (Houston, TX). Protein lysates for western blot were prepared after 24, 48 or 72 hrs of drug treatment at IC70 using RIPA lysis buffer (Thermo Fisher, Waltham, MA) containing protease/phosphatase inhibitors (Roche Diagnostic, Indianapolis, IN). Signals were quantified with NIH ImageJ Imaging Analysis Software or Odyssey Imager software (Li-Cor Bioscience, Lincoln, NE) and normalized to vinculin or alpha-tubulin. Results: Dual combinations of tucatinib with fulvestrant and tucatinib with palbociclib were synergistic in all three HR+/HER2+ cell lines. Addition of a constant dose of fulvestrant to a combination of tucatinib and palbociclib further improved tumor cell killing in all three breast tumor cell lines tested. Combination of tucatinib and palbociclib resulted in greater suppression of phospho-AKT and total and phospho-CDK2 comparing to either agent alone. Treatment with single agents tucatinib and palbociclib lead to a significant increase of cyclin E expression at 48 and 72hrs after treatment, however, this compensatory mechanism of cell cycle progression was not observed after treatment with tucatinib and palbociclib combination. Experiments with the resistant BT474 and MDA-MB-361 subclones showed that cross-treatment of cells resistant to palbociclib with tucatinib and vice versa suppressed cell growth and decreased expression of phospho-ERK1/2, phospho-AKT, phospho-CDK2 and cyclin E. Conclusions: In HR+/HER2+ human breast tumor cell lines combination therapy with tucatinib, palbociclib and fulvestrant demonstrated significant synergy, potentially acting through suppression of phospho-AKT and cyclin E pathways, therefore suggesting a promising novel approach to treatment of HR+/HER2+ breast cancer. Clinical testing of tucatinib, palbociclib and anti-hormonal therapy in patients with HR+/HER2+ metastatic breast cancer is ongoing (phase Ib/II clinical trial NCT03054363). Citation Format: Shagisultanova E, Borakove M, Kabos P, Borges V. Triple targeted combination therapy with tucatinib, palbociclib and fulvestrant is active in hormone receptor and HER2-positive human breast tumor cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-07.

RETRACTED ARTICLE: REST regulates the cell cycle for cardiac development and regeneration

Despite the importance of cardiomyocyte proliferation in cardiac development and regeneration, the mechanisms that promote cardiomyocyte cell cycle remain incompletely understood. RE1 silencing transcription factor (REST) is a transcriptional repressor of neuronal genes. Here we show that REST also regulates the cardiomyocyte cell cycle. REST binds and represses the cell cycle inhibitor gene p21 and is required for mouse cardiac development and regeneration. Rest deletion de-represses p21 and inhibits the cardiomyocyte cell cycle and proliferation in embryonic or regenerating hearts. By contrast, REST overexpression in cultured cardiomyocytes represses p21 and increases proliferation. We further show that p21 knockout rescues cardiomyocyte cell cycle and proliferation defects resulting from Rest deletion. Our study reveals a REST-p21 regulatory axis as a mechanism for cell cycle progression in cardiomyocytes, which might be exploited therapeutically to enhance cardiac regeneration.

Linkage of E2F1 transcriptional network and cell proliferation with respiratory chain activity in breast cancer cells.

Mitochondria are multifunctional organelles; they have been implicated in various aspects of tumorigenesis. In this study, we investigated a novel role of the basal electron transport chain (ETC) activity in cell proliferation by inhibiting mitochondrial replication and transcription (mtR/T) using pharmacological and genetic interventions, which depleted mitochondrial DNA/RNA, thereby inducing ETC deficiency. Interestingly, mtR/T inhibition did not decrease ATP levels despite deficiency in ETC activity in different cell types, including MDA‐MB‐231 breast cancer cells, but it severely impeded cell cycle progression, specifically progression during G2 and/or M phases in the cancer cells. Under these conditions, the expression of a group of cell cycle regulators was downregulated without affecting the growth signaling pathway. Further analysis suggested that the transcriptional network organized by E2F1 was significantly affected because of the downregulation of E2F1 in response to ETC deficiency, which eventually resulted in the suppression of cell proliferation. Thus, in this study, the E2F1‐mediated ETC‐dependent mechanism has emerged as the regulatory mechanism of cell cycle progression. In addition to E2F1, FOXM1 and BMYB were also downregulated, which contributed specifically to the defects in G2 and/or M phase progression. Thus, ETC‐deficient cancer cells lost their growing ability, including their tumorigenic potential in vivo. ETC deficiency abolished the production of reactive oxygen species (ROS) from the mitochondria and a mitochondria‐targeted antioxidant mimicked the deficiency, thereby suggesting that ETC activity signaled through ROS production. In conclusion, this novel coupling between ETC activity and cell cycle progression may be an important mechanism for coordinating cell proliferation and metabolism.

Evidence for asymmetric cell fate and hypoxia induced differentiation in the facultative pathogen protist Colpoda cucullus

Accumulating evidence shows that the evolutionary origin of the asymmetric cell fate and stemness begin in the last eukaryotic ancestor (LECA). The mechanisms of asymmetric division and stemness are conserved in descendants and pathogen protists as Entamoeba invadens and Giardia lamblia. It is to be expected that mechanisms for asymmetric cell fate are also conserved in free-living protists and occasionally facultative pathogens. For a better understanding of the ancestral mechanisms involved and their evolution in the protists, we looked for similar mechanisms of cell cycle progression, growth and differentiation in the facultative pathogen protists Colpoda cucullus. We compare what is known about Colpoda cucullus to our findings in E. invadens.

The protein tyrosine phosphatase SHP-2 is required for EGFRvIII oncogenic transformation in human glioblastoma cells

Oncogenic EGFRvIII is a naturally occurring oncoprotein and is expressed in about 40–50% of human glioblastomas, particularly those that arise de novo. To understand the molecular mechanisms by which this oncoprotein alters transforming phenotypes, and since our previous work indicated that SHP-2 protein tyrosine phosphatase activity modulated EGFRvIII activation and downstream signaling, we examined whether SHP-2 plays a role in EGFRvIII-induced oncogenesis by using both PTEN-deficient U87MG.EGFRvIII and PTEN-intact LN229.EGFRvIII cells. Inhibition of SHP-2 expression by Shp-2 siRNA inhibited cell growth, transformation and altered morphology of these EGFRvIII transformed GBM cells. Ectopic expression of a PTPase-inactive form of SHP-2, SHP-2 C459S, but not its wild-type SHP-2 or either of two SH2 domain mutants, abrogated transformation of EGFRvIII-expressing glioblastomas in soft agar and in nude mice. SHP-2 C459S cells grew slower and exhibited a more flattened morphology with more organized actin stress fibers under both full growth and low serum conditions. Furthermore, shp-2+/− and −/− mouse embryonic fibroblasts (MEFs) could not be transformed by EGFRvIII while shp-2+/+ MEFs displayed a fully transformed phenotype upon introduction of EGFRvIII, again indicating a requirement for functional SHP-2 in EGFRvIII transformation. Moreover, the SHP-2 PTPase activity inhibitor NSC-87877 inhibited endogenous SHP-2 activity, Erk phosphorylation and transformation in both GBM cell lines. EGFRvIII expression recruited SHP-2 to the receptor complex to transduce signals and also increased SHP-2 phosphorylation at Tyr542. Inhibition of EGFRvIII-induced cell growth and transformation by SHP-2 C459S or shp-2 siRNA was mediated by its ability to block cell cycle progression at different phases in these GBM cells. These data indicate that differential activation of SHP-2 phosphorylation at Tyr542 in these two GBM cell lines likely results in increased different PTPase activity and distinct mechanisms of cell cycle progression and SHP-2, in particular its PTPase activity, plays a critical role in EGFRvIII-mediated transformation.

A loss of function screen identifies nine new radiation susceptibility genes

Genomic instability is considered a hallmark of carcinogenesis, and dysfunction of DNA repair and cell cycle regulation in response to DNA damage caused by ionizing radiation are thought to be important factors in the early stages of genomic instability. We performed cell-based functional screening using an RNA interference library targeting 200 genes in human cells. We identified three known and nine new radiation susceptibility genes, eight of which are linked directly or potentially with cell cycle progression. Cell cycle analysis on four of the genes not previously linked to cell cycle progression demonstrated that one, ZDHHC8, was associated with the G2/M checkpoint in response to DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, DNA repair, cell death, cell growth and genomic instability, and to develop new radiation sensitizing agents for radiotherapy.

G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium

Because proliferation of eukaryotic cells requires cell cycle-regulated chromatid separation by the mitotic spindle, it is subject to regulation by mitotic checkpoints. To determine the mechanism of the antiproliferative activity of the flavoprotein-specific inhibitor diphenyleneiodonium (DPI), I have examined its effect on the cell cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. However, unlike the paclitaxel-mediated mitotic block, DPI-treated cells are arrested in the cell cycle prior to mitosis. Although DPI-treated cells can arrest with fully separated centrosomes at opposite sides of the nucleus, these centrosomes fail to assemble mitotic spindle microtubules and they do not accumulate the Thr(288) phosphorylated Aurora-A kinase marker of centrosome maturation. In contrast with paclitaxel-arrested cells, DPI impairs cyclin B1 accumulation. Release from DPI permits an accumulation of cyclin B1 and progression of the cells into mitosis. Conversely, exposure of paclitaxel-arrested mitotic cells to DPI causes a precipitous drop in cyclin B and Thr(288) phosphorylated Aurora-A levels and leads to mitotic catastrophe in a range of cancerous and noncancerous cells. Hence, the antiproliferative activity of DPI reflects a novel inhibitory mechanism of cell cycle progression that can reverse spindle checkpoint-mediated cell cycle arrest.

Human cytomegalovirus pp71: a new viral tool to probe the mechanisms of cell cycle progression and oncogenesis controlled by the retinoblastoma family of tumor suppressors.

The DNA tumor virus oncogenes (adenovirus E1A, simian virus 40 (SV40) large T antigen, and papillomavirus E7) have been instrumental in illuminating the molecules and mechanisms of cell cycle progression and carcinogenesis. However, since these multifunctional proteins target so many important cellular regulators, it is sometimes difficult to establish the functional importance of any individual interaction. Perhaps a herpesvirus protein, newly defined as a cell cycle regulator, can help address these issues. Like the DNA tumor virus proteins, the human cytomegalovirus (HCMV) pp71 protein contains a retinoblastoma protein (Rb) binding motif (LxCxD), and stimulates DNA synthesis in quiescent cells. Unlike E1A, T antigen, and E7, pp71 expression does not induce apoptosis, nor does it cooperate to transform primary cells. Determining how pp71 induces cell cycle progression without invoking apoptosis or leading to cellular transformation may help in defining the signals that ultimately lead to these processes.

Cdk2-dependent Phosphorylation of the NF-Y Transcription Factor and Its Involvement in the p53-p21 Signaling Pathway

Recent studies have suggested that the NF-Y transcription factor is involved in transcription repression of the cell cycle regulatory genes in a response to p53 induction or DNA damage. Here we demonstrate the cdk2-dependent phosphorylation of NF-Y and its involvement in transcription repression by the p53-p21 signaling pathway. Cdk2 phosphorylates two serine residues near the DNA-binding domain of the YA subunit of NF-Y. Cyclin A-cdk2 appears to associate with NF-Y both in vitro and in vivo. Furthermore, YA protein is phosphorylated in parallel with a cell cycle-dependent activation of cdk2 kinase and cyclin A expression. YA phosphorylation is unnecessary for heterotrimer formation with the YB-YC dimer. However, NF-Y containing a phosphorylation-deficient mutant form of YA, YA-aa, has its DNA binding activity impaired. Consistently, YA-aa inhibits transcription activation of a NF-Y target promoter, cdc2, by cdk2. These results facilitate the elucidation of the regulatory mechanisms of cell cycle progression involving the p21-cdk2-NF-Y signaling pathway.

Molecular mechanism of cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I.

The trans-activator protein Tax of human T-cell leukemia virus type I (HTLV-I) plays an important role in the development of adult T-cell leukemia through, at least in part, its ability to stimulate cell growth. We previously reported that Tax induced cell cycle progression from G0/G1 phase to S and G2/M phases in human T-cell line Kit 225 cells. To elucidate molecular mechanism of Tax-induced cell cycle progression, we systematically examined the effects of Tax on biochemical events associated with cell cycle progression. Introduction of Tax into resting Kit 225 cells induced activation of the G1/S transition regulation cascade consisting of activation of cyclin dependent kinase 2 (CDK2) and CDK4, phosphorylation of the Rb family proteins and an increase in free E2F. The kinase activation was found to result from Tax-induced expression of genes for cell cycle regulatory molecules including cyclin D2, cyclin E, E2F1, CDK2, CDK4 and CDK6, and Tax-induced reduction of CDK inhibitors p19(INK4d) and p27(Kip1). These modulations by Tax always paralleled the ability of Tax to activate the NF-kappaB transcription pathway. These results indicate the important role of Tax-mediated trans-activation of the genes for cell cycle regulatory molecules in Tax-induced cell cycle progression.

G Protein βγ Subunits Inhibit Nongenomic Progesterone-induced Signaling and Maturation in Xenopus laevis Oocytes: EVIDENCE FOR A RELEASE OF INHIBITION MECHANISM FOR CELL CYCLE PROGRESSION

Progesterone-induced maturation of Xenopus oocytes is a well known example of nongenomic signaling by steroids; however, little is known about the early signaling events involved in this process. Previous work has suggested that G proteins and G protein-coupled receptors may be involved in progesterone-mediated oocyte maturation as well as in other nongenomic steroid-induced signaling events. To investigate the role of G proteins in nongenomic signaling by progesterone, the effects of modulating Galpha and Gbetagamma levels in Xenopus oocytes on progesterone-induced signaling and maturation were examined. Our results demonstrate that Gbetagamma subunits, rather than Galpha, are the principal mediators of progesterone action in this system. We show that overexpression of Gbetagamma inhibits both progesterone-induced maturation and activation of the MAPK pathway, whereas sequestration of endogenous Gbetagamma subunits enhances progesterone-mediated signaling and maturation. These data are consistent with a model whereby endogenous free Xenopus Gbetagamma subunits constitutively inhibit oocyte maturation. Progesterone may induce maturation by antagonizing this inhibition and therefore allowing cell cycle progression to occur. These studies offer new insight into the early signaling events mediated by progesterone and may be useful in characterizing and identifying the membrane progesterone receptor in oocytes.

Bioprobes for investigating mammalian cell cycle control.

Bioprobes are low molecular weight compounds which are useful for investigating mammalian cell functions. The use of bioprobes has substantially assisted the investigation of complex biochemical processes of the mammalian cell cycle. In this review, cell cycle inhibitors mainly isolated from the microorganism are described and their possibility as an antitumor agents is considered. Most cancer cells have some abnormality in the control mechanism of cell cycle progression. Cyclin-dependent kinases (Cdk), which are activated by the binding with the cyclin and simultaneously by the phosphorylation/dephosphorylation of itself, play important roles as engines in the cell cycle. Tubulins are considered to be one of the most important proteins of the cell division machinery. Therefore, Cdk inhibitors and tubulin binders are possible anticancer drugs. Since the function of proteins controlling the cell cycle is also regulated by phosphorylation and dephosphorylation, inhibitors of protein kinases and phosphatases are considered as possible an antitumor agents. We expect that some bioprobes will be developed for clinical use.

PCNA and total nuclear protein content as markers of cell proliferation in pea tissue

ABSTRACT The identification of cell proliferation markers has been shown to be a useful tool with which to study basic mechanisms of cell cycle progression. The use of immunofluorescence techniques revealed the presence of the proliferating cell nuclear antigen (PCNA) in pea tissue, where we observed a high PCNA expression in proliferating cells of the root meristem compared to noncycling cells of the differentiated leaf. The presence of PCNA was monitored also during the time-course of seed germination, before, during and after the cell cycle resumption of the embryo cells. PCNA is present in embryo cells not only during and after resumption of the cell cycle but also before, when cells have not yet begun replicating their genome. A bivariate flow cytometric analysis of DNA and nuclear protein content was used to localize precisely the cells of the examined pea tissues in different cell cycle phase subcompartments. A high correlation was found between the degree of cell proliferation and the protein content of G1 nuclei, on the one hand, and the percentage of PCNA positive cells on the other.

Hepatocytes: A suitable cell system for studying the control mechanisms of cell cycle progression in mammalians

Hepatocytes: A suitable cell system for studying the control mechanisms of cell cycle progression in mammalians

Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells.

The proliferation of arterial smooth muscle cells (SMCs) plays a critical role in the pathogenesis of arteriosclerosis. Previous studies have indicated that the glycosaminoglycan heparin specifically inhibited the growth of vascular SMCs in vivo and in culture, although the precise mechanism(s) of action have not been elucidated. In this study, we have examined the ability of specific mitogens (PDGF, EGF, heparin-binding growth factors, phorbol esters, and insulin) to stimulate SMC proliferation. Our results indicate that SMCs derived from different species and vascular sources respond differently to these growth factors. We next examined the ability of heparin to inhibit the proliferative responses to these mitogens. In calf aortic SMCs, heparin inhibits a protein kinase C-dependent pathway for mitogenesis. Detailed cell cycle analysis revealed several new features of the effects of heparin on SMCs. For example, heparin has two effects on the Go----S transition: it delays entry into S phase and also reduces the number of cells entering the cycle from Go. Using two separate experimental approaches, we found that heparin must be present during the last 4 h before S phase, suggesting a mid-to-late G1 heparin block. In addition, our data indicate that heparin-treated SMCs, while initially blocked in mid-to-late G1, slowly move back into a quiescent growth state in the continued presence of heparin. These results suggest that heparin may have multiple targets for its antiproliferative effect.